| 51. |
- Petrovic, Dusan, et al.
(författare)
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Challenges and advances in the computational modeling of biological phosphate hydrolysis
- 2018
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Ingår i: Chemical Communications. - 1359-7345 .- 1364-548X. ; 54:25, s. 3077-3089
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Forskningsöversikt (refereegranskat)abstract
- Phosphate ester hydrolysis is fundamental to many life processes, and has been the topic of substantial experimental and computational research effort. However, even the simplest of phosphate esters can be hydrolyzed through multiple possible pathways that can be difficult to distinguish between, either experimentally, or computationally. Therefore, the mechanisms of both the enzymatic and non-enzymatic reactions have been historically controversial. In the present contribution, we highlight a number of technical issues involved in reliably modeling these computationally challenging reactions, as well as proposing potential solutions. We also showcase examples of our own work in this area, discussing both the non-enzymatic reaction in aqueous solution, as well insights obtained from the computational modeling of organophosphate hydrolysis and catalytic promiscuity amongst enzymes that catalyze phosphoryl transfer.
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| 52. |
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| 53. |
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| 54. |
- Petrovic, Dusan, et al.
(författare)
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Molecular modeling of conformational dynamics and its role in enzyme evolution
- 2018
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Ingår i: Current opinion in structural biology. - : Elsevier BV. - 0959-440X .- 1879-033X. ; , s. 50-57
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Tidskriftsartikel (refereegranskat)abstract
- With increasing computational power, biomolecular simulations have become an invaluable tool for understanding enzyme mechanisms and the origins of enzyme catalysis. More recently, computational studies have started to focus on understanding how enzyme activity itself evolves, both in terms of enhancing the native or new activities on existing enzyme scaffolds, or completely de novo on previously non-catalytic scaffolds. In this context, both experiment and molecular modeling provided strong evidence for an important role of conformational dynamics in the evolution of enzyme functions. This contribution will present a brief overview of the current state of the art for computationally exploring enzyme conformational dynamics in enzyme evolution, and, using several showcase studies, illustrate the ways molecular modeling can be used to shed light on how enzyme function evolves, at the most fundamental molecular level.
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| 55. |
- Petrovic, Dusan, et al.
(författare)
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Shuffling Active Site Substate Populations Affects Catalytic Activity : The Case of Glucose Oxidase
- 2017
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Ingår i: ACS Catalysis. - : AMER CHEMICAL SOC. - 2155-5435. ; 7:9, s. 6188-6197
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Tidskriftsartikel (refereegranskat)abstract
- Glucose oxidase has wide applications in the pharmaceutical, chemical, and food industries. Many recent studies have enhanced key properties of this enzyme using directed evolution, yet without being able to reveal why these mutations are actually beneficial. This work presents a synergistic combination of experimental and computational methods, indicating how mutations, even when distant from the active site, positively affect glucose oxidase catalysis. We have determined the crystal structures of glucose oxidase mutants containing molecular oxygen in the active site. The catalytically important His516 residue has been previously shown to be flexible in the wild-type enzyme. The molecular dynamics simulations performed in this work allow us to quantify this floppiness, revealing that His516 exists in two states: catalytic and noncatalytic. The relative populations of these two substates are almost identical in the wild-type enzyme, with His516 readily shuffling between them. In the glucose oxidase mutants, on the other hand, the mutations enrich the catalytic His516 conformation and reduce the flexibility of this residue, leading to an enhancement in their catalytic efficiency. This study stresses the benefit of active site preorganization with respect to enzyme conversion rates by reducing molecular reorientation needs. We further suggest that the computational approach based on Hamiltonian replica exchange molecular dynamics, used in this study, may be a general approach to screening in silico for improved enzyme variants involving flexible catalytic residues.
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| 56. |
- Pfeiffer, Martin, et al.
(författare)
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Essential Functional Interplay of the Catalytic Groups in Acid Phosphatase
- 2022
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Ingår i: ACS Catalysis. - : American Chemical Society (ACS). - 2155-5435. ; 12:6, s. 3357-3370
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Tidskriftsartikel (refereegranskat)abstract
- The cooperative interplay between the functional devices of a preorganized active site is fundamental to enzyme catalysis. An in-depth understanding of this phenomenon is central to elucidating the remarkable efficiency of natural enzymes and provides an essential benchmark for enzyme design and engineering. Here, we study the functional interconnectedness of the catalytic nucleophile (His18) in an acid phosphatase by analyzing the consequences of its replacement with aspartate. We present crystallographic, biochemical, and computational evidence for a conserved mechanistic pathway via a phospho-enzyme intermediate on Asp18. Linear free-energy relationships for phosphoryl transfer from phosphomonoester substrates to His18/Asp18 provide evidence for the cooperative interplay between the nucleophilic and general-acid catalytic groups in the wild-type enzyme, and its substantial loss in the H18D variant. As an isolated factor of phosphatase efficiency, the advantage of a histidine compared to an aspartate nucleophile is similar to 10(4)-fold. Cooperativity with the catalytic acid adds >= 10(2)-fold to that advantage. Empirical valence bond simulations of phosphoryl transfer from glucose 1-phosphate to His and Asp in the enzyme explain the loss of activity of the Asp18 enzyme through a combination of impaired substrate positioning in the Michaelis complex, as well as a shift from early to late protonation of the leaving group in the H18D variant. The evidence presented furthermore suggests that the cooperative nature of catalysis distinguishes the enzymatic reaction from the corresponding reaction in solution and is enabled by the electrostatic preorganization of the active site. Our results reveal sophisticated discrimination in multifunctional catalysis of a highly proficient phosphatase active site.
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| 57. |
- Pinto, Gaspar P., et al.
(författare)
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Exploiting enzyme evolution for computational protein design
- 2022
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Ingår i: TIBS -Trends in Biochemical Sciences. Regular ed.. - : Elsevier. - 0968-0004 .- 1362-4326. ; 47:5, s. 375-389
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Forskningsöversikt (refereegranskat)abstract
- Recent years have seen an explosion of interest in understanding the physicochemical parameters that shape enzyme evolution, as well as substantial advances in computational enzyme design. This review discusses three areas where evolutionary information can be used as part of the design process: (i) using ancestral sequence reconstruction (ASR) to generate new starting points for enzyme design efforts; (ii) learning from how nature uses conformational dynamics in enzyme evolution to mimic this process in silico; and (iii) modular design of enzymes from smaller fragments, again mimicking the process by which nature appears to create new protein folds. Using showcase examples, we highlight the importance of incorporating evolutionary information to continue to push forward the boundaries of enzyme design studies.
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| 58. |
- Purg, Miha, 1988-
(författare)
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Computational Modeling of the Mechanisms and Selectivity of Organophosphate Hydrolases
- 2018
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Computational modeling is becoming an increasingly integral part of (bio)chemistry, providing a powerful complementary view into the dynamics, binding, and reactivity of biochemical systems. In particular, molecular simulations based on multiscale models are now regularly employed in studies of enzymatic reactions, offering invaluable mechanistic insight through the lens of molecular energy landscapes. In this thesis, I used the empirical valence bond (EVB) and related methods to study the mechanisms and selectivity of organophosphate hydrolases.Organophosphate hydrolases are a diverse class of enzymes capable of degrading some of the most toxic compounds known to mankind, including pesticides and chemical warfare agents. They are particularly interesting from a mechanistic and evolutionary point of view, having evolved the ability to catalyze the hydrolysis of compounds which were introduced to nature less than a century ago. Moreover, they show promise as effective organophosphate decontamination agents and a thorough understanding of their function is fundamental to the future design of efficient and selective biocatalysts. As organophosphate hydrolases are metal-dependent enzymes, a reliable metal model was a prerequisite to our simulations. First, I present the development of force-field independent parameters for several alkaline-earth and transition-metal ions described using the nonbonded cationic dummy model. The model was subsequently employed in EVB simulations to probe the origin of metal-ion activity and selectivity patterns observed in methyl parathion hydrolase (MPH) and to provide mechanistic insight into its paraoxonase and promiscuous arylesterase activities. I further set out to resolve open mechanistic questions surrounding diisopropyl fluorophosphatase (DFPase) by performing extensive simulations of two mechanistic pathways proposed in literature, including calculating the effects of mutations, temperature, and protonation states on the rate of hydrolysis. Using this knowledge, I address the origin of cross-selectivity between DFPase and a structurally similar enzyme serum paraoxonase 1 (PON1). Finally, I present the latest developments in the software used to perform the simulations.
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| 59. |
- Purg, Miha, et al.
(författare)
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Empirical Valence Bond Simulations of Organophosphate Hydrolysis : Theory and Practice
- 2018
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Ingår i: PHOSPHATASES. - : ELSEVIER ACADEMIC PRESS INC. - 9780128138816 ; , s. 3-51
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Bokkapitel (refereegranskat)abstract
- Recent years have seen an explosion of interest in understanding the mechanisms of phosphate ester hydrolysis in biological systems, using a range of computational approaches, each with different advantages and limitations. In this contribution, we present the empirical valence bond (EVB) approach as a powerful tool for modeling biochemical reactivity, using the example of organophosphate hydrolysis by diisopropyl fluorophosphatase as our model reaction. We walk the reader through the protocol for setting up and performing EVB simulations, as well as key technical considerations that need to be taken into account. Finally, we provide examples of the applications of the EVB approach to understanding different experimental observables.
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| 60. |
- Purg, Miha, et al.
(författare)
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Similar Active Sites and Mechanisms Do Not Lead to Cross-Promiscuity in Organophosphate Hydrolysis : Implications for Biotherapeutic Engineering
- 2017
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Ingår i: Journal of the American Chemical Society. - : AMER CHEMICAL SOC. - 0002-7863 .- 1520-5126. ; 139:48, s. 17533-17546
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Tidskriftsartikel (refereegranskat)abstract
- Organophosphate hydrolases are proficient catalysts of the breakdown of neurotoxic organophosphates and have great potential as both biotherapeutics for treating acute organophosphate toxicity and as bioremediation agents. However, proficient organophosphatases such as serum paraoxonase 1 (PON1) and the organophosphate-hydrolyzing lactonase SsoPox are unable to hydrolyze bulkyorganophosphates with challenging leaving groups such as diisopropyl fluorophosphate (DFP) or venomous agent X, creating a major challenge for enzyme design. Curiously, despite their mutually exclusive substrate specificities, PON1 and diisopropyl fluorophosphatase (DFPase) have essentially identical active sites and tertiary structures. In the present work, we use empirical valence bond simulations to probe the catalytic mechanism of DFPase as well as temperature, pH, and mutational effects, demonstrating that DFPase and PON1 also likely utilize identical catalytic mechanisms to hydrolyze their respective substrates. However, detailed examination of both static structures and dynamical simulations demonstrates subtle but significant differences in the electrostatic properties and solvent penetration of the two active sites and, most critically, the role of residues that make no direct contact with either substrate in acting as "specificity switches" between the two enzymes. Specifically, we demonstrate that key residues that are structurally and functionally critical for the paraoxonase activity of PON1 prevent it from being able to hydrolyze DFP with its fluoride leaving group. These insights expand our understanding of the drivers of the evolution of divergent substrate specificity in enzymes with identical active sites and guide the future design of organophosphate hydrolases that hydrolyze compounds with challenging leaving groups.
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| 61. |
- Risso, Valeria A., et al.
(författare)
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De novo active sites for resurrected Precambrian enzymes
- 2017
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Ingår i: Nature Communications. - : NATURE PUBLISHING GROUP. - 2041-1723. ; 8
-
Tidskriftsartikel (refereegranskat)abstract
- Protein engineering studies often suggest the emergence of completely new enzyme functionalities to be highly improbable. However, enzymes likely catalysed many different reactions already in the last universal common ancestor. Mechanisms for the emergence of completely new active sites must therefore either plausibly exist or at least have existed at the primordial protein stage. Here, we use resurrected Precambrian proteins as scaffolds for protein engineering and demonstrate that a new active site can be generated through a single hydrophobic-to-ionizable amino acid replacement that generates a partially buried group with perturbed physico-chemical properties. We provide experimental and computational evidence that conformational flexibility can assist the emergence and subsequent evolution of new active sites by improving substrate and transition-state binding, through the sampling of many potentially productive conformations. Our results suggest a mechanism for the emergence of primordial enzymes and highlight the potential of ancestral reconstruction as a tool for protein engineering.
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| 62. |
- Romero-Rivera, Adrian, et al.
(författare)
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Complex Loop Dynamics Underpin Activity, Specificity, and Evolvability in the (beta alpha)(8) Barrel Enzymes of Histidine and Tryptophan Biosynthesi
- 2022
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Ingår i: JACS Au. - : American Chemical Society (ACS). - 2691-3704. ; 2:4, s. 943-960
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Tidskriftsartikel (refereegranskat)abstract
- Enzymes are conformationally dynamic, and their dynamical properties play an important role in regulating their specificity and evolvability. In this context, substantial attention has been paid to the role of ligand-gated conformational changes in enzyme catalysis; however, such studies have focused on tremendously proficient enzymes such as triosephosphate isomerase and orotidine 5'-monophosphate decarboxylase, where the rapid (mu s timescale) motion of a single loop dominates the transition between catalytically inactive and active conformations. In contrast, the (beta alpha)(8)-barrels of tryptophan and histidine biosynthesis, such as the specialist isomerase enzymes HisA and TrpF, and the bifunctional isomerase PriA, are decorated by multiple long loops that undergo conformational transitions on the ms (or slower) timescale. Studying the interdependent motions of multiple slow loops, and their role in catalysis, poses a significant computational challenge. This work combines conventional and enhanced molecular dynamics simulations with empirical valence bond simulations to provide rich details of the conformational behavior of the catalytic loops in HisA, PriA, and TrpF, and the role of their plasticity in facilitating bifunctionality in PriA and evolved HisA variants. In addition, we demonstrate that, similar to other enzymes activated by ligand-gated conformational changes, loops 3 and 4 of HisA and PriA act as gripper loops, facilitating the isomerization of the large bulky substrate ProFAR, albeit now on much slower timescales. This hints at convergent evolution on these different (beta alpha)(8)-barrel scaffolds. Finally, our work reemphasizes the potential of engineering loop dynamics as a tool to artificially manipulate the catalytic repertoire of TIM-barrel proteins.
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| 63. |
- Szeler, Klaudia
(författare)
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Computational Protein Evolution : Modeling the Selectivity and Promiscuity of Engineered Enzymes
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Enzymes are biological catalysts that significantly increase the rate of all biochemical reactions that take place within cells and are essential to maintain life. Many questions regarding their function remain unknown. Experimental techniques, such as kinetic measurements, spectroscopy, and site-directed mutagenesis, can provide relevant information about enzyme structure, key residues, active site conformations, and kinetics. However, they struggle to provide a full picture of enzyme catalysis. Combining experiments with computational techniques gives the possibility to generate a complete explanation with atomistic resolution. Computational modeling offers an incredibly robust toolkit that can provide detailed insight into the reactivity and dynamics of biomolecules.Compounds that contain phosphate and sulfate groups are essential in the living world. They are present as i.e., a biological source of energy (ATP), signaling molecules (GTP), coenzymes, building blocks (DNA, RNA). Furthermore, phosphate esters can be used as insecticides, herbicides, flame retardants, and as chemical weapons. Cleavage of the phosphate bond involves an extremely low rate of spontaneous hydrolysis, nevertheless it is common reaction in living organisms. Phosphatases (enzymes catalysing cleavage of phosphate bond) are crucial in both physiological regulation as well as serious pathological conditions including asthma, immunosuppression, cardiovascular diseases, diabetes.Understanding the basis of phosphoryl and sulfuryl transfer reactions is crucial for medical, biological, and biotechnological industries in order to i.e., create and improve existing drugs, modify enzyme structures, understand the development of some diseases. However, despite decades of both experimental and computational studies, mechanistic details of these reactions remain controversial. These reactions can occur via multiple different mechanisms involving intermediate steps or transition state structures. To solve these puzzles, we performed computational studies to verify the reaction pathway of diaryl sulfate diesters hydrolysis. We suggest that the reaction proceeds through a concerted mechanism with a loose (slightly dissociative) transition state.Serum paraoxonase 1 (PON1) is calcium-dependent lactonase, which is bound to high-density lipoprotein (HDL) with apolipoprotein A-I (ApoA-I). The enzyme is highly promiscuous and catalyzes the hydrolysis of multiple, different types of chemical compounds, such as lactones, aromatic esters, oxons, and organophosphates. We performed several, complex studies on PON1’s reaction mechanism, promiscuity, PON1-HDL interactions, and evolutionary trajectories. One of the most extensively used approaches in this thesis was the empirical valence bond (EVB) method. Our models reproduce essential experimental observables and provide mechanistic insights and a better understanding of the enzymes role and its evolutionary derived promiscuity.
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| 64. |
- Tatum, Natalie J., et al.
(författare)
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Relative Binding Energies Predict Crystallographic Binding Modes of Ethionamide Booster Lead Compounds
- 2019
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Ingår i: The Journal of Physical Chemistry Letters. - : AMER CHEMICAL SOC. - 1948-7185. ; 10:9, s. 2244-2249
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Tidskriftsartikel (refereegranskat)abstract
- Transcriptional repressor EthR from Mycobacterium tuberculosis is a valuable target for antibiotic booster drugs. We previously reported a virtual screening campaign to identify EthR inhibitors for development. Two ligand binding orientations were often proposed, though only the top scoring pose was utilized for filtering of the large data set. We obtained biophysically validated hits, some of which yielded complex crystal structures. In some cases, the crystallized binding mode and top scoring mode agree, while for others an alternate ligand binding orientation was found. In this contribution, we combine rigid docking, molecular dynamics simulations, and the linear interaction energy method to calculate binding free energies and derive relative binding energies for a number of EthR inhibitors in both modes. This strategy allowed us to correctly predict the most favorable orientation. Therefore, this widely applicable approach will be suitable to triage multiple binding modes within EthR and other potential drug targets with similar characteristics.
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| 65. |
- Uduwela, Dimanthi R., et al.
(författare)
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Enhancing the Steroid Sulfatase Activity of the Arylsulfatase from Pseudomonas aeruginosa
- 2018
-
Ingår i: ACS Catalysis. - : AMER CHEMICAL SOC. - 2155-5435. ; 8:9, s. 8902-8914
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Tidskriftsartikel (refereegranskat)abstract
- Steroidal sulfate esters play a central role in many physiological processes. They serve as the reservoir for endogenous sex hormones and form a significant fraction of the steroid metabolite pool. The analysis of steroid sulfates is thus essential in fields such as medical science and sports drug testing. Although the direct detection of steroid sulfates can be readily achieved using liquid chromatography-mass spectrometry, many analytical approaches, including gas chromatography-mass spectrometry, are hampered due to the lack of suitable enzymatic or chemical methods for sulfate ester hydrolysis prior to analysis. Enhanced methods of steroid sulfate hydrolysis would expand analytical possibilities for the study of these widely occurring metabolites. The arylsulfatase from Pseudomonas aeruginosa (PaS) is a purified enzyme capable of hydrolyzing steroid sulfates. However, this enzyme requires improvement to hydrolytic activity and substrate scope in order to be useful in analytical applications. These improvements were sought by applying semirational design to mutate amino acid residues neighboring the enzyme active site. Mutagenesis was implemented on both single and multiple residue sites. Screening by ultra-high performance liquid chromatography-mass spectrometry was performed to test the steroid sulfate hydrolysis activity of these mutant libraries against testosterone sulfate. This approach revealed the steroid sulfate binding pocket and resulted in three mutants that showed an improvement in catalytic efficiency (V-max/K-M) of more than 150 times that of wild-type PaS. The substrate scope of PaS was expanded, and a modest increase in thermostability was observed. Finally, molecular dynamics simulations of enzyme-substrate complexes were used to provide qualitative insight into the structural origin of the observed effects.
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| 66. |
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| 67. |
- Yang, Gloria, et al.
(författare)
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Higher-order epistasis shapes the fitness landscape of a xenobiotic-degrading enzyme
- 2019
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Ingår i: Nature Chemical Biology. - : NATURE PUBLISHING GROUP. - 1552-4450 .- 1552-4469. ; 15:11, s. 1120-1128
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Tidskriftsartikel (refereegranskat)abstract
- Characterizing the adaptive landscapes that encompass the emergence of novel enzyme functions can provide molecular insights into both enzymatic and evolutionary mechanisms. Here, we combine ancestral protein reconstruction with biochemical, structural and mutational analyses to characterize the functional evolution of methyl-parathion hydrolase (MPH), an organophosphate-degrading enzyme. We identify five mutations that are necessary and sufficient for the evolution of MPH from an ancestral dihydrocoumarin hydrolase. In-depth analyses of the adaptive landscapes encompassing this evolutionary transition revealed that the mutations form a complex interaction network, defined in part by higher-order epistasis, that constrained the adaptive pathways available. By also characterizing the adaptive landscapes in terms of their functional activities towards three additional organophosphate substrates, we reveal that subtle differences in the polarity of the substrate substituents drastically alter the network of epistatic interactions. Our work suggests that the mutations function collectively to enable substrate recognition via subtle structural repositioning.
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| 68. |
- Zhan, Shaoqi, et al.
(författare)
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Capturing the Role of Explicit Solvent in the Dimerization of Ru-V(bda) Water Oxidation Catalysts
- 2017
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Ingår i: Angewandte Chemie International Edition. - : Wiley-VCH Verlagsgesellschaft. - 1433-7851 .- 1521-3773. ; 56:24, s. 6962-6965
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Tidskriftsartikel (refereegranskat)abstract
- A ground-breaking empirical valence bond study for a soluble transition-metal complex is presented. The full reaction of catalyst monomers approaching and reacting in the Ru-V oxidation state were studied. Analysis of the solvation shell in the reactant and along the reaction coordinate revealed that the oxo itself is hydrophobic, which adds a significant driving force to form the dimer. The effect of the solvent on the reaction between the prereactive dimer and the product was small. The solvent seems to lower the barrier for the isoquinoline (isoq) complex while it is increased for pyridines. By comparing the reaction in the gas phase and solution, the proposed p-stacking interaction of the isoq ligands is found to be entirely driven by the water medium.
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